Extracorporeal clearance of organophosphates from blood on an acoustic separation device

ABSTRACT

Systems, methods, and compositions for removing organophosphate toxins from blood are disclosed herein. The compositions include a lipid-based capture particle that displays BChE that binds to the toxin. The methods include acoustically separating toxins bound to lipid-based capture particles from blood factors of whole blood.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/168,822, now issued as U.S. Pat. No. 9,504,780, which claims priorityto U.S. Application No. 61/758,495 filed Jan. 30, 2013. The entirecontents of the application listed above are hereby incorporated byreference in their entirety.

BACKGROUND

Extracorporeal clearance of organophosphates (OP) from the blood is atechnique to counter toxicity. Used as an adjuvant therapy, clearance ofOP toxins makes other current therapeutic for clearance of OP toxins(for example, oximes or plasma administration) more effective. The mostwidely used method of extracorporeal OP clearance is hemodialysis, whichis typically only effective with fat-insoluble toxins. Clearance offat-soluble OP toxins, like parathion, or its active in vivo metaboliteparaoxon, requires hemoperfusion over adsorbent surfaces like activatedcharcoal or activated resins. The resins used to remove fat-solubletoxins, e.g., paraoxon, can also non-specifically adsorb or damage bloodfactors (e.g., platelets, red blood cells, white blood cells, etc.),which can lead to clinical complications such as loss of platelets,leukopenia, hypocalcaemia, hypoglycemia, and fibrinogen reduction. Theloss of blood factors can lead to systemic bleeding, which can causedeath in patients receiving this treatment.

SUMMARY

In one aspect, the present technology provides devices for removingorganophosphates from whole blood including: a thermoplasticmicrofluidic separation channel having an upstream end and downstreamend, wherein the separation channel comprises: 1) a first inletconfigured to introduce flowing whole blood into a proximal end of theseparation channel, wherein the whole blood comprises plasma, bloodfactors, and organophosphates; 2) a first outlet at the downstream endof the separation channel positioned substantially along thelongitudinal axis of the separation channel; and 3) a second outlet atthe downstream end positioned adjacent a first wall of the separationchannel; and the device includes an acoustic transducer positionedadjacent to the separation channel for imposing a standing acoustic wavetransverse to the flow of blood through a particle migration region ofthe separation channel; and a lipid-based capture particle injectorcontaining lipid-based capture particles and configured to introducelipid-based capture particles into the whole blood before the bloodreaches the particle migration region of the separation channel, whereinthe lipid-based capture particle comprises a first population of lipids,and silicon oil; wherein the first population of lipids comprisesorganophosphate affinity molecules linked to the lipids of firstpopulation of lipids. In some implementations, lipid-based particle alsoincludes a second population of lipids, wherein the second population oflipids form a lipid layer in which the first population is embedded.

In some implementations, the lipid-based capture particles are in theform of a liposome, vesicle, emulsion, lipid encapsulated droplet, orcombinations thereof. In some implementations, the lipid-based captureparticle is a liposome.

In some implementations, the organophosphate affinity molecule is BChE.

In some implementations, the organophosphate affinity molecules arelinked to the first population of lipids via PEG.

In some implementations, the silicone oil is encapsulated within thelipid-based particle.

In some implementations, the device also includes a reservoir in fluidiccommunication with the lipid-based capture particle injector.

In some implementations, the separation channel comprises walls having athickness at a particle aggregation point that is a multiple of onequarter of the wavelength of an acoustic wave acting on the walls of theseparation channel.

In another aspect, the present technology provides methods of cleansingblood of a subject including: flowing whole blood into an inlet of amicrofluidic separation channel wherein the whole blood comprisesplasma, blood factors, and organophosphates; introducing lipid-basedcapture particles into the whole blood which bind to theorganophosphates, wherein the lipid-based capture particle comprises afirst population of lipids, a second population of lipids, silicon oiland cholesterol; wherein the first population of lipids comprises anorganophosphate affinity molecule linked to the lipids of the firstpopulation of lipids; and applying a standing acoustic wave transverseto a direction of flow of the whole blood through the separation channelsuch that the blood factors aggregate to about the axial center of theseparation channel and the lipid-based capture particles with boundorganophosphates aggregate along at least one wall of the separationchannel. In some implementations, the methods also include cycling offthe standing acoustic wave such that the duty cycle of the standingacoustic wave is between about 75% and about 95%.

In some implementations, the methods also include collecting bloodfactors of the whole blood at a first outlet positioned at a downstreamend of the separation channel at about the axial center of theseparation channel.

In some implementations, the methods also include collecting lipid-basedcapture particles through at least a second outlet positioned at thedownstream end of the separation channel adjacent to the at least onewall along which the lipid-based capture particles are aggregated.

In some implementations, the lipid-based capture particle also include asecond population of lipids, wherein the second population of lipidsform a lipid layer in which the first population is embedded. In someimplementations, the lipid-based capture particles have an oppositecontrast factor than the blood factors.

In some implementations, the lipid-based capture particles are betweenabout 10 μm and 20 μm in diameter.

In some implementations, the methods also include reintroducing theblood factors back into the subject after flowing the whole bloodthrough the microfluidic separation channel.

In another aspect, the present technology provides compositionsincluding organophosphate affinity molecules, a first population oflipids and silicon oil, wherein the first population of lipids comprisesorganophosphates affinity molecules linked to the lipids of the firstpopulation of lipid, wherein the silicone oil is encapsulated within thelipid-based capture particle, and the at least one organophosphateaffinity molecule is displayed on the surface of the lipid-based captureparticle.

In some implementations, the organophosphate affinity molecule is BChE.

In some implementations, the first population of lipids is selected fromDSPE, DPPE, DMPE, or a combination thereof.

In some implementations, the lipid-based capture particle also includesa second population of lipids, wherein the second population of lipidsform a lipid layer in which the first population is embedded. In someimplementations, the second population of lipids is selected from DOPC,DOPG, DOPE, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the described implementations may be shownexaggerated or enlarged to facilitate an understanding of the describedimplementations. In the drawings, like reference characters generallyrefer to like features, functionally similar and/or structurally similarelements throughout the various drawings. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the teachings. The drawings are not intended to limitthe scope of the present teachings in any way. The system and method maybe better understood from the following illustrative description withreference to the following drawings in which:

FIG. 1 is a non-limiting, exemplary block diagram of a system forremoving organophosphates from a patient.

FIG. 2 is a non-limiting, exemplary top view of a single-stageseparation channel, such as can be used in the system of FIG. 1.

FIG. 3 is a non-limiting, exemplary top view of a network of two-stageseparation channels, such as can be used in FIG. 1.

FIG. 4 is a non-limiting, exemplary cross sectional view of asingle-stage separation channel, such as the separation channel of FIG.2, mounted to a bulk transducer.

FIG. 5A is a non-limiting, exemplary cross sectional view a single-stageseparation channel, as depicted in FIG. 2, containing a plurality ofparticles lacking an active acoustic transducer.

FIG. 5B is a non-limiting, exemplary cross sectional of a single-stageseparation channel, as depicted in FIG. 2, containing a plurality ofparticles adjacent to an active acoustic transducer.

FIG. 6A is a non-limiting, exemplary top view of a separation channel,as depicted in FIG. 2, in which fluid flows through the channel withoutthe application of the standing acoustic wave.

FIG. 6B is a non-limiting, exemplary top view of a separation channel,as depicted in FIG. 2, after the application of a standing acousticwave.

FIG. 7 is a non-limiting, exemplary cut away of a lipid-based captureparticle.

FIGS. 8A-E are non-limiting, exemplary illustrations of the componentsincluded in a lipid-based capture particle, as depicted in FIG. 7.

FIG. 9 is a non-limiting, exemplary flow chart of a method for cleansingblood with a two-stage separation channel, as depicted in FIG. 3.

FIG. 10 is a non-limiting, exemplary flow chart of a method forcleansing blood with a single-stage separation channel, as depicted inFIG. 2.

DETAILED DESCRIPTION

The various concepts introduced above and discussed in greater detailbelow may be implemented in any of numerous ways, as the describedconcepts are not limited to any particular manner of implementation.Examples of specific implementations and applications are providedprimarily for illustrative purposes.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. For example, reference to “a cell” includesa combination of two or more cells, and the like.

As used herein, “organophosphate affinity molecule” (“OP affinitymolecule”) refers to a molecule that binds to organophosphates. By wayof example, but not by limitation, butyrylcholinesterase (BChE) is anorganophosphate affinity molecule that binds to parathion and/orparaoxon, which are organophosphates.

As used herein, “blood factors” refers to elements in the blood requiredfor the normal function of blood. Whole blood includes, but is notlimited to, plasma and blood factors. Blood factors include, but are notlimited to, platelets, red blood cells, and white blood cells. By way ofexample, but not by limitations, loss of normal blood function can becaused by a loss of, for example, platelets, leukopenia, hypocalcaemia,hypoglycemia, and fibrinogen reduction.

As used herein, “lipid-based capture particle(s)” refers to acomposition including a first population of lipids, silicone oil, and atleast one organophosphate affinity molecule which is displayed on thesurface of the lipid-based capture particle. In some implementations,the lipid-based capture particles include cholesterol. In someimplementations, the first population of lipids is linked to at leastone organophosphate affinity molecule via a PEG linker. In someimplementations, the lipid-based capture particle further comprises asecond population of lipids, wherein the second population of lipidsprovide a lipid layer in which the first population of lipids isembedded. Examples of organophosphate affinity molecules include, butare not limited to, butyrylcholinesterase (BChE). In someimplementations, the lipid-based capture particles are in the form ofliposomes, vesicles, emulsions, lipid encapsulated droplets orcombinations thereof.

Generally, the present technology relates to compositions, systems,devices, and methods for removing organophosphates (OP), e.g., parathionand/or paraoxon, from whole blood. Parathion is a fat-soluble OPpesticide. Paraoxon is the active metabolite of parathion.

In various implementations, the disclosure relates to removing OP, e.g.,parathion and/or paraoxon, from whole blood by acoustically separatingOP from the blood via high throughput microfluidic arrays. In someimplementations, lipid-based capture particles displaying at least oneorganophosphate affinity molecule are introduced and mixed with theblood, ex vivo, to form complexes with the OP, yielding complexes thatare effectively and efficiently separated from the blood factors andplasma by acoustic separation. The lipid-based capture particlestypically bind to the OP.

The lipid-based capture particles can be administered to whole blood ofanimals, e.g., humans, that have been exposed to OP, e.g., parathion, orsuspected of being exposed to OP to facilitate the removal of OP fromthe blood.

Lipid-Based Capture Particles

In some implementations, the lipid-based capture particles of thepresent technology are in the form of liposomes vesicles, emulsions,lipid encapsulated droplets or combinations thereof. In someimplementations, a lipid-based capture particle is in the form of aliposome. In some implementations, the liposome includes a firstpopulation of lipids and a second population of lipids, wherein thesecond population of lipids provide a lipid layer in which the firstpopulation of lipids is embedded. In some implementations, the firstpopulation of lipids is linked to at least one organophosphate affinitymolecule. Examples of organophosphate affinity molecule include, but arenot limited to, butyrylcholinesterase (BChE) molecule. BChE is anon-specific cholinesterase enzyme that hydrolyses many differentcholine esters. BChE can bind to parathion and/or paraoxon. In someimplementations, the first population of lipids is linked to BChE.

In some implementations, the organophosphate affinity molecule, e.g.,BChE, is linked to the lipid (e.g., the first population of lipids) viaa PEG linker to form an organophosphate affinity molecule-PEG-lipidcomplex, e.g., BChE-PEG-lipid. In some implementations, a firstpopulation of lipids is linked to a plurality of PEG molecules to formone or more PEG-lipids. In some implementations, the PEG-lipid is linkedto BChE. In some implementations, the PEG is linked to the BChE, and thePEG-BChE is linked to the lipid. In some implementation the lipidscomprising the first population of lipids includes, but is not limitedto, synthetic, semi-synthetic, or naturally occurring lipids. By way ofexample, but not by way of limitation, in some implementations, thelipids comprising the first population of lipids is one or more of1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE),N-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), andN-(Carbonyl-methoxypolyethyleneglycol2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE).

In some implementations, at least one organophosphate affinitymolecule-PEG-lipid complex, e.g., BChE-PEG-lipid, is combined with asecond population of lipids to form a lipid-based capture particle,e.g., a liposome. In some implementations, the second population oflipids provide a lipid layer, e.g., a lipid-bilayer, in which the firstpopulation of lipids is embedded. In some implementations, the secondpopulation of lipids are the same lipid. In some implementations, thesecond population of lipids comprises a mixture of different lipids. Insome implementations, the lipids of the first population and the secondpopulation are the same. In some implementations, the lipids of thefirst population and the second population are different. In someimplementations, the second population of lipids includes but is notlimited to one or more of synthetic, semi-synthetic, or naturallyoccurring lipids. By way of example, but not by way of limitation, thesecond population of lipids includes one or more of1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

In some implementations, the lipid-based capture particle also includessilicone oil. In some implementations, the silicone oil is encapsulatedwithin the lipid-based capture particle. In some implementations, theviscosity of the silicone oil is about 100, about 200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000centistokes, or ranges between any two of these values. In anotherimplementation, the viscosity of the silicone oil is between about 1000to 10,000, or between about 2000 to 9000, or between about 3000 to 8000,or between about 4000 to 7000, or between about 5000 to 6000centistokes.

Additionally or alternatively, in some implementation, the liposome ofthe lipid-based capture particle includes cholesterol. In someimplementations, the lipid mixture includes between about 0.01 to 1 mol%, between about 1 to 5 mol %, between about 5 to 40 mol %, betweenabout 10 to 35 mol %, between about 15 to 30 mol %, or between about 20to about 25 mol % of cholesterol.

Additionally or alternatively, in some implementation, the liposome ofthe lipid-based capture particle also includes one or more activemolecules. An active molecule is incorporated into the liposome for itseffect on the liposome density and acoustic activity. Example of activemolecule includes, but is not limited to, per-fluoro-butane andultrasound imaging contrast agents.

Methods for Making the Lipid-Based Capture Particles

Methods for Linking Organophosphate Affinity Molecule to a Lipid

In some implementation, organophosphate affinity molecule, e.g., BChE,is linked to PEG. In some implementations, BChE is presented as amixture containing monomers, dimers, and tetramers of BChE. In someimplementations, PEG is mixed with the BChE mixture at about an 80:1ratio, which produces 1:1 ratio of PEG to BChE monomeric unit with highpercentage of dimers being produced.

In some implementations, PEG is linked to N-Hydroxy succinimide (NHS).In some implementations, the NHS-PEG is linked to the organophosphateaffinity molecule. In some implementations, the NHS-PEG is linked toBChE. In some implementation, the PEG in NHS-PEG links to lysineresidues or the N-terminus of BChE. In some implementations, aNHS-activated PEG is linked to a phospholipid, i.e., NHS-PEG-DSPE. Insome implementations, the NHS-PEG-DSPE is linked to BChE, i.e.,BChE-PEG-phospholipid.

In another implementation, N-terminal thiol groups are targeted byactivated PEG-o-pyridylthioester. In some implementations, N-terminalthiol groups targeting is performed after the PEG-o-pyridylthioester islinked to a lipid. In some implementations, cysteine-functionalizedphospholipids are linked to PEG, e.g., Cys-PEG-DSPE,

In yet another implementation, PEG functionalized phospholipids arelinked to sialic acid sugars. Sialic acid sugar facilitates conjugationof glycans on BChE. In some implementations, the PEG functionalizedphospholipid is linked to at least one BChE. PEG functionalizedphospholipids include, but are not limited to, DSPE-PEG-NH₂ andDSPE-PEG-COOH.

In some implementations, conjugation of BChE to PEG-phospholipidsincludes the dissolution of an activated PEG functionalized lipid, e.g.,DSPE-PEG-NH₂, in an organic solvent such as 1:2 ratio of DMSO/methanol,the addition of BChE and coupling reagent EDC. In some implementations,the reaction can be monitored by thin layer chromatography (TLC) usingKMnO₄ or p-anisaldehyde. In some implementations, the un-reactedlipid-PEG can be extracted by gel electrophoresis.

In some implementations, about 10 to 50-fold molar excess offunctionalized PEG-lipid, e.g., NHS-PEG-DSPE, is combined withorganophosphate affinity molecules to produce organophosphate affinitymolecules-PEG-lipid complexes, e.g., BChE-PEG-DSPE. In someimplementations, the organophosphate affinity molecules e.g., BChE, arefirst dissolved in a conjugation buffer (e.g., sodium bicarbonate 100 mMbuffer, pH 8.5 or other amine-free buffer at pH 7-8.5). In someimplementations, the functionalized PEG-lipid are added next at a finalconcentration of at least 10 mg/ml. Additionally, or alternatively, insome implementations, the functionalized PEG-lipid are added next at afinal concentration of 10 to 50-fold molar excess. In someimplementations, the mixture is mixed for about 30 to 60 minutes at roomtemperature. In another implementation, the mixture is mixed for about 2hours at 4° C.

In some implementations, the further purification ofBChE-PEG-phospholipid is performed by size exclusion chromatography(SEC). SEC can be performed using Sepharose or Superdex™ 200, which isnot only useful for purification of the protein product but also mapsthe location of the PEG-bound sites within the protein by trypsindigestion.

In some implementations, the purity of the BChE-PEG-phospholipid isdetermined. Purity can be determined by commonly used methods in the artincluding, but not limited to, SDS-PAGE, mass spectroscopy, and HPLC.

In some implementations, the location or the site of the PEG attachmentto the BChE is identified by peptide mapping followed by LC/MS orLC/MS/MS analysis.

Method for Making Affinity Molecule Displaying Lipid-Based CaptureParticles

In some implementations, the conjugated BChE-PEG-lipids, describedabove, are incorporated into a lipid-based capture particle.

In some implementations, at least one BChE-PEG-lipid is combined with amixture of lipids, e.g., a second population of lipids, to form alipid-based capture particle. In some implementations, the secondpopulation of lipids provide a lipid layer, e.g., a lipid-bilayer, inwhich the first population of lipids is embedded. In someimplementation, the mixture of lipids, e.g., a second population oflipids, includes cholesterol. In some implementations, silicone oil withdifferent viscosity is employed in different formulations to furtherenhance the flow efficiency of the lipid-based capture particles throughthe device. In some implementations, active molecules, such asper-fluoro-butane, are incorporated for their effect on the lipid-basedparticle's density and acoustic activity.

In some implementations, the mixture of lipids, e.g., a secondpopulation of lipids, includes, but is not limited to, synthetic,semi-synthetic, or naturally occurring lipids. In some implementations,the mixture of lipids is selected from1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG) and1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

In some implementations, the lipid-based capture particle also includessilicone oil. In some implementations, the silicone oil is encapsulatedwithin the lipid-based capture particle. In some implementations, theviscosity of the silicone oil is about 100, about 200, about 300, about400, about 500, about 600, about 700, about 800, about 900, about 1000centistokes, or ranges between any two of these values. In anotherimplementation, the viscosity of the silicone oil is between about 1000to 10,000, or between about 2000 to 9000, or between about 3000 to 8000,or between about 4000 to 7000, or between about 5000 to 6000centistokes.

Additionally or alternatively, in some implementation, the lipid-basedcapture particle also includes cholesterol. In some implementations, thelipid mixture includes between about 5 to 40 mol %, or between about 10to 35 mol %, or between about 15 to 30 mol %, or between about 20 toabout 25 mol % of cholesterol.

In some implementations, the lipid-based particle comprises betweenabout 1 to 30 mol % of BChE-PEG-lipids, between about 5 to 40 mol % ofcholesterol, between about 5 to 20 mol % of silicone oil, and betweenabout 50 to 85 mol % of the mixture of lipids.

In some implementations, the size and monodispersity of the lipid-basedcapture particles are controlled by modified extrusion techniques, anddialysis/diafiltration. In some implementations, film rehydrationtechnique to provide large vesicles with diameter of 15-30 μm. In someimplementations, hydration of the lipid film in warm (35° C.) waterfollowed by addition of re-hydrated buffer to pre-hydrated lipid film.In some implementations, the re-hydrated lipid-buffer solution isincubated for several hours at a temperature above lipid phasetransition temperature, e.g., >10° C. In some implementations, thelipid-based capture particles swell and strip away from a Teflon surfaceproducing small cloud of lipid-based capture particles that can beharvested by a Pasteur Pipette.

The formation of liposomes, vesicles, emulsions, and lipid encapsulateddroplets is known in the art, and the compositions and methods disclosedherein are not intended to be limited by the method of formation ofthese structures. By way of example, but not by way of limitation, knownmethods may be used to form the lipid-based particles disclosed herein;additionally or alternatively, the methods described herein may also beused.

In some implementations, dynamic light scattering (DLS) and transmissionelectron microscopy (TEM) is used to determine the final size anddispersion of the lipid-based capture particles. In someimplementations, lipid ratio in the lipid-based capture particles isdetermined by analysis such as, but limited to, HPLC, GPC, and/orMALDI-MS. In some implementations, the extent of BChE-PEG-lipidconjugation is monitored by incorporation of fluorescent dye (e.g.,Calcein) in the lipid-based capture particle and measuring the amount ofBChE before and after Calcein leakage by spectrophotometer.

In some implementations, BChE enzyme activity in lipid-based captureparticles is assayed by a modified Ellman activity assay to ensure BChEretained its enzymatic activity. The assay detects the activity of BChEby measuring the formation of a substrate hydrolysis product thatabsorbs at 412 nm.

In some implementations, the ability of the lipid-based capture particleto clear organophosphates, e.g., parathion and/or paraoxon, from bloodis determined in vitro. In some implementations, up to five differentconcentrations of the lipid-based capture particle is added to bloodthat has been contaminated with excess paraoxon. In someimplementations, the blood is incubated at 37° C. for one hour andfiltered using a commercially available filter plate (molecular weightcutoff ultrafiltration) to remove parathion and/or paraoxon that isbound to the BChE in the blood. In some implementations, theconcentration of unbound parathion and/or paraoxon in the blood filtrateis determined by GC-MS.

In some implementations, after incubation at 37° C. for one hour, thelipid-based capture particles bound with organophosphates, e.g.,parathion and/or paraoxon, is separated from the blood via an acousticdevice and system as described below. In some implementations, the bloodis pumped into at least one channel of a microfluidic devices. In someimplementations, the blood is subjected to an acoustic wave in thechannel. In some implementations, the acoustic wave separates thelipid-based capture particles bound with organophosphates, e.g.,parathion and/or paraoxon, from the blood factors and the lipid-basedcapture particles bound with parathion and/or paraoxon selectivelyremoved.

Devices and Systems

FIG. 1 illustrates a system 100 for removing organophosphates, e.g.,parathion and/or paraoxon, from the blood. In the system 100,organophosphate contaminated blood is removed from a patient via anintravenous line 102. The blood is then pumped to a mixing chamber 104by a first pump 103. In the mixing chamber 104, lipid-based captureparticles are mixed with whole blood. In some implementations, thecomponents of the lipid-based capture particles are stored in areservoir 115. In some implementations, the lipid-based captureparticles are formed and are stored in the reservoir 115. From thereservoir 115, the lipid-based capture particles are pumped by a secondpump 106 into the mixing chamber. In some implementations, thelipid-based capture particles are formed as the contents of thereservoir 115 are extruded from a micronozzle 105 at the entrance to themixing chamber 104. In the mixing chamber 1-4, the affinity molecules onthe lipid-based capture particles, e.g., BChE, bind to theorganophosphates, e.g., parathion and/or paraoxon. From the mixingchamber 104, the whole blood and lipid-based capture particles enter amanifold system 107. The manifold system 107 distributes the whole bloodand lipid-based capture particles to a plurality of separation channelscontained within the microfluidic flow chamber 108. The microfluidicflow chamber 108 is in contact with or in functional proximity, e.g.,sits atop, at least one bulk piezoelectric acoustic transducer 109. Theacoustic waves generated by the bulk piezoelectric acoustic transducersare used to funnel the contents of the whole blood and lipid-basedcapture particles with bound organophosphates to specific outlets of theseparation channels. As the whole blood flows through the microfluidicflow chamber 108, cleansed blood flows to a first outlet 110. Afterexiting the first outlet 100, the cleansed blood returns to the patient101, via a second intravenous line 111. The lipid-based captureparticles and other waste material removed from the blood exit themicrofluidic flow chamber 108 via a second outlet 112. Next, the wastematerial and lipid-based capture particles enter a waste collection unit113. In the waste collection unit 113, the lipid-based capture particlesare separated from the waste material. Once separated, the wastematerial is discarded and the lipid-based capture particles are returnedto the reservoir 107 by tubing 114. Once returned to the reservoir 107,the lipid-based capture particles are reused in the system to removeadditional waste material from whole blood as it continues to flowthrough the system.

The system 100, as illustrated, includes a pump 103 for moving bloodfrom the patient 101 to the mixing chamber 104. In some implementations,the pump operates continuously, while in other implementations the pumpworks intermittently, and only activates when the level of whole bloodin the mixing chamber 104 or manifold falls below a set threshold. Insome implementations, the flow rate of the pump is configurable, suchthat the rate the blood exits the patient can be configured to be fasteror slower than if no pump was used. In yet other implementations, noexternal pump is required. In this example, the blood is transported tothe mixing chamber 104 by the pressure generated by the patient's ownheart. In some implementations, the patient 101 is connected to a bloodpressure monitor, which in turn controls the pump. Example pumps caninclude, but are not limited, to peristaltic pumps or any other pumpsuitable for flowing blood.

As illustrated in the system 100, lipid-based capture particles are alsopumped into the mixing chamber. A second pump 106 pumps the ingredientsto form the lipid-based capture particles from a reservoir 107 to themixing chamber 104. In some implementations, the components of thelipid-based capture particles are continuously agitated in the reservoir107 in order to keep the components well mixed. The components areformed into lipid-based capture particles as they enter the mixingchamber 104. The components enter the mixing chamber 104 through amicronozzle 105. In some implementations, the lipid-based captureparticles are formed in the reservoir 107. In some implementations, themicronozzle 105 injects the lipid-based capture particles into themixing chamber 104. In other implementations, the micronozzle 105injects the lipid-based capture particles into the manifold system 107,and in yet other implementations the micronozzle 105 is positioned suchthat it injects lipid-based capture particles directly into theseparation channels of the microfluidic chamber 108. In someimplementations, the micronozzle 105 is a micro-machined nozzle,configured to allow a specific amount of the lipid-based captureparticle components through the nozzle at a given time. In someimplementations, the micronozzle is an array of micronozzles. In yetother implementations, the micronozzle is a membrane with pores. In someimplementations, the pump 106 is configured to flow the contents of thereservoir through the micronozzle 105 at a predetermined rate such thatthe amphipathic characteristics of the molecules of the components ofthe captures particles cause the lipid-based capture particles tospontaneously form as they exit the micronozzle 105.

In some implementations, a micronozzle is not used to generate thelipid-based capture particles. In these implementations, the lipid-basedcapture particles are premade. The lipid-based capture particles arethen stored in the reservoir and then introduced into the system by thepump 106 at either the mixing chamber 104, manifold system 107, and/orthe separation channels of the microfluidic flow chamber 108.

As illustrated in system 100, the whole blood containingorganophosphates, e.g., parathion and/or paraoxon, and the lipid-basedcapture particles enter the mixing chamber 104. In some implantations,the contents of the mixing chamber are continuously agitated to improvedistribution of the lipid-based capture particles throughout the wholeblood and organophosphates such that the lipid-based capture particlesbind to the organophosphates. In some implementations, anticoagulants orblood thinners are introduced into the mixing chamber 104 to assist theblood as it flows through the system 100. In some implementations, themixing chamber 104 contains a heating element for warming the contentsof the mixing chamber 104.

The contents of the mixing chamber 104 then flow into the manifoldsystem 107, as illustrated by system 100. The manifold system 107 flowsthe whole blood, organophosphates, e.g., parathion and/or paraoxon, andlipid-based capture particles into the inlets of the plurality ofseparation channels of the microfluidic flow chamber 108.

In the illustrated system 100, the microfluidic flow chamber 108contains a plurality of separation channels. The lipid-based captureparticles and organophosphates, are driven with standing acoustic wavesto outlets. In some implementations, the separation occurs during asingle stage, while in other implementations, the separation occurs overa plurality of stages. In some implementations, the microfluidic flowchamber is disposable.

As show in the illustrations of system 100, the microfluidic flowchamber 108 sits atop a bulk piezoelectric acoustic transducer 109. Insome implementations, the system 100 contains a single bulkpiezoelectric acoustic transducer 109, while in other implementationsthe system 100 contains a plurality of bulk piezoelectric acoustictransducers 109.

In some implementations, the bulk piezoelectric acoustic transducer 109is glued to the microfluidic flow chamber 108. In other implementationsthe microfluidic flow chamber 108 is clamped to the bulk piezoelectricacoustic transducer 109 so the microfluidic flow chamber may easily beremoved from the system. In other implementations the adhesive materialconnecting the bulk piezoelectric acoustic transducer 109 to themicrofluidic flow chamber 108 is removable, for example by heating theadhesive.

The bulk piezoelectric acoustic transducer 109 imposes a standingacoustic wave on the separation channels of the microfluidic flowchamber 108 transverse to the flow of the fluid within the microfluidicflow chamber 108. The standing acoustic waves are used to drive fluidconstituents towards or away from the walls of the separation channelsor other aggregation axes.

In some implementations, the dimensions of the separation channels areselected based on the wavelength of the imposed standing wave such apressure node exists at about the center or other interior axis of theseparating channel, while antinodes exists at about the walls of theseparation channel. Particles are driven to different positions withinthe channel based on the sign of their acoustic contrast factor at arate that is proportional to the magnitude of their contrast factor.Particles with a positive contrast factor (e.g., blood factors) aredriven towards the pressure node within the interior of the separationchannel. In contrast, particles with a negative contrast factor aredriven toward the pressure antinodes. These principles are depicted anddescribed further in relation to FIGS. 5A and 5B.

Based on these principles, blood factors can be separated fromlipid-based capture particles with bound organophosphates, e.g.,parathion and/or paraoxon, in two ways. In one way, as described furtherin relation to FIGS. 2 and 10, lipid-based capture particles with boundorganophosphates are selected to have negative contrast factors, whichis opposite to the positive contrast factors of the blood factors. Thus,in response to the standing acoustic wave, the blood factors are driventowards the resulting pressure node while the lipid-based captureparticles with bound organophosphates are driven towards the antinodes.

This technique can be used in a single-stage separation system. As wholeblood, organophosphates, e.g., parathion and/or paraoxon, andlipid-based capture particles mix in the mixing chamber 104 and continueto mix as flowing through the manifold system 107, the lipid-basedcapture particles bind to the parathion and/or paraoxon. As the wholeblood, organophosphates, and lipid-based capture particles enter thearea of the separation channel where the standing acoustic wave isimparted, the standing acoustic wave drives the lipid-based captureparticles and bound organophosphates to a specific axis (e.g., againstthe wall of the separation channel) and the blood factors of the wholeblood to a second axis (e.g., the middle of the separation channel).Thus, the lipid-based capture particles bound with organophosphates canbe collected from the edges of the separation channel and disposed ofwhile the cleaned blood is collected and returned to the patient.

Alternatively, lipid-based capture particles can be separated fromformed blood factors based on a time of flight principle. That is, ifthe lipid-based capture particles are selected to have a contrast factorthat is the same sign as that of the blood factors, but with asubstantially different magnitude, and assuming the blood factors andlipid-based capture particles are substantially aligned prior to theapplication of a standing wave at a distance away from the positivepressure node induced by the wave, the blood factors and lipid-basedcapture particles will migrate towards the pressure node at differentrates. Thus, the blood factors and lipid-based capture particles can becollected separately at a point where the higher contrast factorparticles (lipid-based capture particles or blood factors depending onthe selected lipid-based capture particles) have move sufficiently farfrom the initial aggregation axis that they have separated from thelipid-based capture particles due to their difference in acoustophoreticmobility. Thus, in some implementations, a two-stage separation processis employed. In the two-stage process, blood factors and lipid-basedcapture particles are first aggregated along a common first axis of theseparation channel using a first standing acoustic wave. Then after theyhave reached the common aggregation axis, a second standing acousticwave drives the blood factors and lipid-based capture particles to asecond aggregation axis. However, instead of waiting until the bloodfactors and lipid-based capture particles all reach the secondaggregation axis, the channel splits to direct the particles having thelower acoustophoretic mobility down a first outlet. The particles thathave a greater acoustophoretic mobility, which would have alreadymigrated towards the second aggregation axis to a point that they arebeyond entrance to the first outlet, flow out a second outlet. Thisseparation technique is described further in relation to FIGS. 3 and 10.

As illustrated in the system 100, the cleansed blood exits themicrofluidic flow chamber 108 at a first outlet 110. From there theblood is returned to the patient 101 via an intravenous supply line 111.In some implementations, the blood in the supply line 111 is reheated tobody temperature before returning to the patient 101. In otherimplementations an infusion pump is used to return the blood to thepatient 101, while in the system 100 the pressure generated in thesystem by pumps 103 and 106 is adequate to force the blood to return tothe patient 101.

As illustrated in the system 100, waste material (e.g., the lipid-basedcapture particle bound with parathion and/or paraoxon) exit themicrofluidic flow chamber 108 and enter a waste collection unit 113. Insome implementations, the waste collection unit 113 contains alipid-based capture particle recycler. The lipid-based capture particlerecycler unbinds the parathion and/or paraoxon from the lipid-basedcapture particles. The lipid-based capture particles are then returnedto the reservoir 107 via tubing 114. The organophosphates, e.g.,parathion and/or paraoxon, are then disposed of. In someimplementations, the organophosphates are saved for further testing.

While the system 100 is described above for the in-line cleansing of apatient's blood, in alternative implementations, the system 100 can beused to cleanse stored blood. For example, the system 100 can be used tocleanse collected blood for later infusion to help ensure the safety ofthe blood.

FIG. 2 illustrates an example single-stage separation channel suitablefor use within the microfluidic flow chamber 108 of the blood cleansingsystem 100. The separation channel includes an inlet 202, a flow channel203, a first outlet 204, a first outlet channel 206, a second outletchannel 207, and a second outlet 205. The separation channel ismanufactured in a sheet of material 201.

In FIG. 2, whole blood, parathion and/or paraoxon, and lipid-basedcapture particles enter the separation channel at the inlet 202 from themanifold system 107. The whole blood, organophosphates, e.g., parathionand/or paraoxon, and lipid-based capture particles then flow the lengthof the flow channel 203. The flow channel is subdivided into threeregions: an upstream region, a downstream region, and a migrationregion. The migration region lies between the upstream and downstreamregions, and is the region of the flow channel where the standingacoustic wave is imparted transverse to the flow of particles. As theblood factors of the whole blood, lipid-based capture particles and theorganophosphates, enter the migration region, the standing acoustic wavedrives the lipid-based capture particles bound to the organophosphates,e.g., parathion and/or paraoxon, to the side walls of the separationchannel, and the blood factors of the whole blood to the center of thechannel. The blood factors of the whole blood then exit the separationchannel through the outlet 204 located at about the central axis of theseparation channel. The lipid-based capture particles andorganophosphates, then exit the separation channel through the first andsecond outlet channels 206 and 207, which terminates in the secondoutlet 205. In some implementations, the blood factors are driven to thewalls of the separation channel and the lipid-based capture particleswith bound organophosphates remain in the center of the separationchannel.

In some implementations, the separation channel 200 can separateorganophosphates, e.g., parathion and/or paraoxon, from any fluid. Asdiscussed above and later in relation to FIGS. 5 and 7, the separationchannel 200 can be used to remove parathion and/or paraoxon from anyfluid. For example, the separation channel 200 may be used to removeorganophosphates, e.g., parathion and/or paraoxon, from, but not limitedto, blood plasma, blood serum, water, and lymph.

In the implementation of FIG. 2, the outlet 205 is formed from themerging of two outlet channels 206 and 207. In some implementations, thestreams do not rejoin, but lead to separate outlet terminals.

In FIG. 2, the particles are separated in the same plane as the sheet ofmaterial 201 (i.e., particles are aligned to the left, right, or centerof the channel); however, in other implementations, the particles areseparated out of plane. For example, in some implementations, theparticles are aligned with the top, middle, or bottom of the channel.

In FIG. 2, the sheet of material 201 can include, but is not limited to,polystyrene, glass, polyimide, acrylic, polysulfone, thermoplastic, andsilicon. The channel can be manufactured by a number of manufacturingtechniques, including, but not limited to, milling, embossing, andetching. In some embodiments, the channels are microchannels ofpolystyrene. In some embodiments, the channels are microchannels ofthermoplastic.

In some implementations, the channels are made from 1 mm thickconventional polystyrene sheets, and seal using adhesivelessthermocompression bonding at 95° C. In some implementations, thechannels are made from machining and bonding methods, see Tsao, C. andDeVoe, D., Microfluidics and Nanofluidics, 6(1): 1-16 (2009), thatresult in post-bond dimensional accuracy to within 10 μm and bonds thatwithstand up to 300 kPa internal pressure.

In some implementations, a higher frequency standing acoustic wave canbe applied to create two pressure nodes within the separation channel200, both spaced apart from the sidewalls of the channel and separatedby an anti-node. In one such implementation, the blood factors in theblood aggregate into two substantially parallel streams near thesidewalls along the pressure nodes, while the lipid-based captureparticles migrate to the center of the channel in line with theanti-node. In such implementations, the lipid-based capture particlesexit the separation channel through the outlet 204, while the bloodexits the separation channel through the first and second outletchannels 206 and 207.

FIG. 3 illustrates an example network 300 of multistage separationchannels suitable for use the blood cleansing system 100 depicted inFIG. 1. The network of separation channels includes a plurality of firstinlets 301. FIG. 3 also includes first and second acoustic bulktransducers 302 and 305, respectively. Additionally, each separationchannel includes an upstream outlet 303 and a second inlet 304. Theupstream outlet 303 of each channel is connected to the second inlet 304of its neighboring channel. The fluid exits the separation channelsthrough a first downstream outlet 307 or second downstream outlet 306.

In each separation channel of the network 300, the flow channel throughwhich most of the fluid in the channel flows shifts after the upstreamoutlet 303. The portion of the separation channel prior to the shift isreferred to as the upstream portion 308 and the portion after the shiftis referred to as the downstream portion 309. As the particles withinthe blood continue to flow down the separation channel, one subset ofparticles is driven into a first downstream outlet 307 and a secondsubset of the particles are driven into a second downstream outlet 306.For example, the cleaned blood exits through the first downstream outlet307 and the lipid-based capture particles with bound organophosphates,e.g., parathion and/or paraoxon, exits through the second downstreamoutlet 306.

As described above, and illustrated in FIG. 3, the separation channelsin the network 300 are generally divided into upstream portions 308 anddownstream portions 309. The two portions are distinguished by a shiftin the separation channel. The angle of the shift is referred to as thebranching angle. The branching angle for a particular implementation ischosen to substantially align a wall of the downstream portion 309 withan interior axis of the upstream portion 308. The selected axiscorresponds to the location of a pressure node induced in the channel bythe first acoustic transducer 302. For example, in some implementations,a wall of the downstream portion 309 is configured to align with aninterior axis substantially in the middle of the upstream portion 308.

In the illustration of FIG. 3, there is a plurality of first inlets 301.The number of first inlets, and thus separation channels, can beincreased to n where n is the number of separation channels required tomeet the flow demands of a specific implementation. In someimplementations, the first inlets 301 are configured to accept flowingwhole blood and a plurality of lipid-based capture particles. Theflowing whole blood includes plasma, a plurality of blood factors and aplurality of OP molecules, such as, e.g., parathion and/or paraoxon. Theblood factors of blood can include leukocytes (white blood cells),erythrocytes (red blood cells), and thrombocytes (platelets). In someimplementations, the lipid-based capture particles begin binding to theOP (e.g., parathion and/or paraoxon) as they are mixed as they flow downthe length of the upstream portion 308. In other implementations, thelipid-based capture particles are mixed with the whole blood prior toflowing through the first inlet 301, and thus the binding of thelipid-based capture particles and the OP (e.g., parathion and/orparaoxon) can begin before the blood is flowed through the first inlet301.

As the fluid in the separation channels flows downstream it passes overa first bulk acoustic transducer 302 and eventually a second bulkacoustic transducer 305. The bulk acoustic transducers 302 and 305impart standing acoustic waves on the separation channels. The standingacoustic waves are transverse to the flow of fluid through theseparation channels. In some implementations, the acoustic bulktransducers 302 and 305 emit standing acoustic waves of differentwavelengths. In other implementations the acoustic bulk transducers 302and 305 emit standing acoustic waves of the same wavelength. Exampleacoustic waves can have, but are not limited to, wavelengths betweenabout 1.0 and about 4.0 MHz, or between about 1.5 and about 3.5 MHz, orbetween about 2.0 and about 3.0 MHz.

In the network 300, the first acoustic bulk 302 transducer aligns theblood factors, lipid-based capture particles, and parathion and/orparaoxon in the interior of the upstream portions 308 of the separationchannels. After the particles in the fluid are aligned in the middle ofthe flow channels, the channels angle to align the particles with wallsof the downstream portions 309. With all particles aligned in the middleof the flow channel, fluid substantially away from the middle is free oflipid-based capture particles and OP, e.g., parathion and/or paraoxon,Thus, at the angle in the flow channels, a portion of the fluid,substantially void of blood factors, lipid-based capture particles andOP, exits through the upstream outlets 303. The particles continue toflow downstream, now substantially aligned with a wall of the respectivedownstream portions 309. Fluid entering the downstream portions 309 ofthe separation channels from the second inlets 304 ensures continuedflow of the blood factors and lipid-based capture particles through thedownstream portions of the 309 of the separation channels.

In the second stage particles are separated based on the speed at which(and thus distance) they travel from a given aggregation axis towards asecond pressure node induced in the channel by a second standingacoustic wave. As indicated above, due to the angling of the separationchannels, the blood factors and lipid-based capture particles enter thedownstream portions of the separation channels aggregated along onewall.

As the particles flow along the wall of the separation flow channels,they pass over a second bulk transducer 305, which emits a secondstanding acoustic wave transverse to the flow of the particles. Thesecond standing acoustic wave drives the particles away from the wall.Based on the magnitude of their contrast factor, a first subset ofparticles (e.g., the blood factors of blood) moves away from the wall ata faster rate than a second subset of particles (e.g., the lipid-basedcapture particles bound with parathion and/or paraoxon).

This differential rate of movement is achieved by using lipid-basedcapture particles that have an acoustophoretic mobility that issubstantially different from that of the blood factors of blood. Thedifferent acoustophoretic mobility is in turn based on the magnitude ofthe contrast factor of lipid-based capture particles being substantiallydifferent from the magnitude of the contrast factor of the blood factorsof blood. The speed at which the two groups move away from the wall canbe calculated, and the rate of flow of blood through the system isknown. Thus, the distance the blood factors will be moved away from thewall of a separation channel at a given location after being exposed tothe second acoustic standing wave can also be calculated. This distanceis termed d(f,x), where d is the distance traveled away from the wallgiven a specific fluid flow rate (f) and a specific distance (x) afterthe application of the standing acoustic wave. Based on thiscalculation, the separation channel can be divided into two outlets. Thesecond downstream outlet 306 is positioned along the wall the bloodfactors and lipid-based capture particles were previously flowing. Thesecond downstream outlet 306 is constructed to have a width just smallerthan d(f, x). Thus, the blood factors, having traveled a distance ofd(f,x) away from the wall due to the second standing acoustic wave wouldhave been driven beyond the second outlet by the time they reach thedistance (x), and thus are driven into the first downstream outlet 307.In contrast, the lipid-based capture particles bound to the parathionand/or paraoxon, having traveled a distance substantially less thand(f,x) due to their lesser acoustophoretic mobility, remainsubstantially near the wall of the downstream portion 309 and exit thesecond outlet 306. In some implementations, the distance (x) traveledbetween exposure to the standing acoustic wave and entering the firstdownstream outlet 307 is between about 1 and about 10 cm.

FIG. 4 is an illustrative cross-section of a separation channel 400similar to the separation channel depicted in FIG. 2. The separationchannel 400 includes a top layer 401 sitting atop a bottom layer 402. Achannel is created in the bottom layer 402. When the top layer 401 isplaced on the bottom layer 402 a lumen 403 is created. The separationchannel 400 sits atop a bulk piezoelectric transducer 404. Theseparation channel 400 is secured to the bulk transducer 404, by acoupling adhesive 405 and/or mechanical clamp. In some implementations,the coupling adhesive is cyanoacrylate glue.

The bottom layer 402 and top layer 401 of the separation channel 400 aremanufactured from a substrate sheet. The substrate sheet can be made of,without limitation, polystyrene, glass and polyimide, polyacrylic,polysulfone, thermoplastic, and silicon. In some implementations, thebottom layer 402 is manufactured by milling, embossing, and/or etching.After creating the two layers, they can be joined together bythermacompression, mechanical clamping, adhesive bonding, and/or plasmabonding. As described above, the separation channel sits atop anacoustic bulk transducer 404. The transducer 404 imparts a standingacoustic wave of a specific wavelength (

) across the channel. The dimensions of the bottom layer 402, top layer401, and lumen 403 are dependent on the selected wavelength. The widthof the lumen 403 is equal to about half the wavelength (

_(fluid)/2) of the acoustic wave in the fluid. The thickness of the sidewall is equal to about a multiple of one quarter of the wavelength (n×

_(wall)/4) of the acoustic wave in the solid channel wall. The height ofthe lumen is preferably less than one quarter of the wavelength (<

_(fluid)/4) of the acoustic wave in the fluid, and the thickness of thetop layer 401 can be arbitrarily selected; however, in someimplementations is chosen to be greater than one quarter of thewavelength (>

_(wall)/4) of the acoustic wave in the solid channel wall.

In some other implementations, the bottom layer 402, top layer 401, andlumen 403 can have different relative dimensions. For separationchannels 400 formed from a thermoplastic, such as polystyrene,polyimide, polyacrylic, or polysulfone, the width of the lumen 403 inthe bottom layer 402 is less than the one-half the wavelength of theacoustic wave in the fluid. In some implementations, the width of thelumen 403 in a thermoplastic separation channel 400 is between aboutone-fourth and three-eighths the wavelength of the acoustic wave in thefluid (i.e., about 25%-50% narrower than the half the wavelength, assuggested above). The shorter width results from the smaller impedancemismatch between the thermoplastic walls of the separation channel andthe fluid passed through the channel. This lower mismatch providesimperfect acoustic reflection, thereby motivating the narrower channel.Particularly in comparison to glass or silicon-based separationchannels, thermoplastic separation channels are substantially lessexpensive to manufacture.

In one implementation, a separation channel formed from polystyrene canoperate with an acoustic wave having a 1.0 and about 4.0 MHz, or betweenabout 1.5 and about 3.5 MHz, or between about 2.0 and about 3.0 MHz.Assuming the channel is configured for carrying water, the lumen of theseparation channel may be about 0.4 mm wide, or about 40% narrower thanhalf the wavelength of the wave. Moreover, the sidewalls of the bottomlayer 402 of a thermoplastic-based separation channel may besignificantly wider than sidewalls formed from materials that serve asbetter acoustic reflectors.

In some implementations, the channel cross section is a shallowrectangle with its width, w, matched to one half the wavelength, λ, ofsound in the fluid (i.e., the blood plasma). In some implementations,the wavelength depends on the chosen frequency, f, and is obtained fromreference tables of acoustic velocity, c, according to the relationλ=c/f. In some implementations, the nominal channel width is set tow=c/(λf), in order to achieve a resonant standing half-wave across thefluid-filled channel. In some implementations, the acoustic focusing isbetween about 1.0 and about 4.0 MHz, or between about 1.5 and about 3.5MHz, or between about 2.0 and about 3.0 MHz. In some implementations,the channel widths are in the range of about 0.2 to 0.8 mm.

FIGS. 5A and 5B are cross sectional views of particles suspended in afluid as they flow through a separation channel similar to theseparation channel 200. For FIGS. 5A and 5B, the flow of the fluid istransverse to the plane of the drawings. In some implementations, thefluid is whole blood, and the particles are the blood factors andlipid-based capture particles. For illustrative purposes, FIGS. 5A and5B contains two particles, red blood cells (white dots) and lipid-basedcapture particles (black dots). FIG. 5A illustrates blood flowingthrough a channel without a standing acoustic wave being imparted on thechannel and its contents. In FIG. 5A, the particles remain homogenouslymixed throughout the channel. In FIG. 5B, a standing wave is imparted onthe channel. The standing acoustic wave 501 creates two node types. Apressure node occurs at 502. The node extends across the full height ofthe lumen. The channel dimensions set forth above in relation to FIG. 4yield a pressure node at approximately the center of the channel.

Particles are aligned based on the sign of their contrast factor.Particles with a positive contrast factor (e.g., the blood factors ofblood) are driven towards a pressure node 502. In contrast, particleswith a negative contrast factor (e.g., lipid-based capture particlesused in the single-stage device described above) are driven toward thepressure antinodes 503.

FIG. 6A is a top view of a separation channel 600, as depicted in FIG.2, in which fluid is flown through the separation channel 600 withoutthe application of the standing acoustic wave. The separation channel600 includes three outlets 602, 603 and 604. As with FIG. 5A, particlessuspended in the fluid are homogeneously distributed throughout thefluid, and thus are not readily discernible in the image. The particlesflow substantially evenly out of all three outlets 602, 603 and 604.

In contrast, FIG. 6B is a top view of the separation channel 600, asdepicted in FIG. 2, after the application of a standing acoustic wave,according to one illustrative embodiment.

In FIG. 6B, as a result of the standing acoustic wave, the particles 601suspended in the fluid is aligned with the middle of the separationchannel 600. Once aligned with the middle of the separation channel 600,the particles 601 exit the separation channel 600 through the middleoutlet 602. The remaining fluid, substantially devoid of particles,exits the separation channel through the side outlets 603 and 604.

FIG. 7 is an illustrative example of a lipid-based capture particle 700in the form of a liposome. The lipid-based capture particle 700 includesa lipid bilayer 701 encapsulating a fluid 702. Displayed on the surfaceof the lipid bilayer are BChE molecules 703. The BChE molecules bind andliposome capture parathion and/or paraoxon 704 and or other OP toxins.

More specifically, the lipid bilayer 701 forms a lipid-based captureparticle. In some implementations, the lipids may be, but are notlimited to 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or a combinationthereof. The lipid-based capture particle is tuned for acousticallyinduced mobility. Entities that differ in size, density, and/orcompressibility have the greatest differential mobility in acousticfields and thus are the most readily separable. Therefore, in someimplementations, the size, density, and/or compressibility of thelipid-based capture particles is modified to distinguish the lipid-basedcapture particle from the blood factors of blood. In someimplementations, the acoustic mobility of a particle is proportional toits volume. For example, in some implementations, the lipid-basedcapture particles are between about 2 μm to 35 μm, or between about 5 μmto 30 μm, or between about 10 μm to 25 μm, or between about 15 μm to 20μm in diameter. In some implementations, use of such larger lipid-basedcapture particles results in a more distinct separation between thelipid-based capture particles and red blood cells.

In implementations that adjust the compressibility of the lipid-basedcapture particle, the rigidity of the lipid-based capture particle canbe adjusted by controlling the lipid components in lipid-based captureparticle. The length and saturation of the lipid hydrocarbon tail,cross-linking of the hydrophobic domains, and/or the inclusion ofcholesterol, and/or PEG will all affect the fluidity and compressibilityof a lipid-based capture particle.

In other implementations, the density of the lipid-based captureparticle, e.g., in the form of a liposome, is engineered byencapsulating an acoustically active fluid 702. In these implementationsthe acoustical active molecule can be an ultra sound imaging contrastagent, glycerine, castor oil, coconut oil, paraffin, air, and/orsilicone oil.

In other implementations, all of the above described characteristics aremanipulated to create a lipid-based capture particle with the greatestpossible difference in contrast factor compared to a formed element.

As described above, the acoustically induced mobility of a particle isbased on the contrast factor of the particle. For a lipid basedlipid-based capture particle, the contrast factor is dominated by theproperties of the encapsulated fluid. The contrast factor is based onthe bulk modulus (K) and density (

) of the encapsulated fluid. When suspended in blood, the contrastfactor (

) for a lipid-based capture particle, encapsulating a specific fluid, iscalculated with the below equation:

$\phi = {\frac{{5\rho} - {2 \cdot 1.02}}{{2\rho} + 1.02} + \frac{2.2}{K}}$

Table 1 provides the

, K, and then calculated

-factor based on the above equation.

TABLE 1 Calculated Contrast Factors Materials

 (g/ml) K(Gpa)

Encapsulated glycerine 1.25 4.7 +0.73 Fluids castor oil 1.03 2.06 −0.06coconut oil 0.92 1.75 −0.36 paraffin 0.80 1.66 −0.58 silicone oil 1.041.09 −1.00 air 0.002 1.4 −3.55 Blood factors white blood cell 1.02 2.5+0.12 red blood cell 1.10 3.0 +0.34

In some implementations, such as the implementation of FIG. 3, thelipid-based capture particles have a contrast factor that is lower inmagnitude, but still of the same sign as the blood factors. In theseimplementations, the low contrast factor of the lipid-based captureparticles can be achieved by making the lipid-based capture particlessufficiently small to reduce their contrast factor to below that of theblood factors.

FIG. 8 illustrates an overview of the process of making and using alipid-based capture particle. The OP affinity molecules (e.g., BChEmolecules for purposes of this illustrative implementation) of FIG. 8Aare embedded in the lipid-based capture particle, (e.g., a liposome forpurposes of this illustrative implementation) of FIG. 8B to produce BChElipid-based capture particles as illustrated in FIG. 8C. Next, thelipid-based capture particles are combined with a blood or other fluidcontaining OP, e.g., parathion and/or paraoxon as illustrated in FIG.8D. The parathion and/or paraoxon then bind to the lipid-based captureparticles. FIG. 8E illustrates bound parathion and/or paraoxon can thenbe removed from the fluid by acoustically moving the lipid-based captureparticles whereas parathion and/or paraoxon are not removed from thefluid.

FIG. 9 is a flow chart of a method 900 to cleanse blood of OPs such asparathion and/or paraoxon in a blood cleaning separation channel similarto the two-stage device described in FIG. 3. First whole blood is flowedthrough a microfluidic separation channel (step 901). Lipid-basedcapture particles are introduced into the whole blood (step 902). Then,blood factors of the blood and the lipid-based capture particles aredirected away from the walls of the separation channel (step 903). Next,the lipid-based capture particles and blood factors are directedalongside a wall of the separation channel (step 904). Then, bloodfactors are driven away from the walls of the separation channel (step905). Finally, the blood factors are collected in a first downstreamoutlet and the lipid-based capture particles are collected in a seconddownstream outlet (steps 906 and 907, respectively).

Referring to FIGS. 3 and 9, the method of cleansing blood includesflowing whole blood through a microfluidic separation channel (step901). The whole blood contains plasma; a plurality of blood factors suchas red blood cells, white blood cells, and platelets; and OP molecules,such as, e.g., parathion and/or paraoxon. In some implementations, thewhole blood is flowed through a plurality of microfluidic separationchannels, such as the network of channels 300, connected to one anotherby a manifold system, while in other implementations a single separationchannel is used. In some implementations, the whole blood is extractedfrom a patient. In other implementations the whole blood is collectedfrom a patient or donor and stored prior to cleansing.

Lipid-based capture particles are introduced into the whole blood (step902). In some implementations, the lipid-based capture particles areintroduced into the whole blood at the first inlet 301 of the separationchannel depicted in FIG. 3. In other implementations, the lipid-basedcapture particles are introduced into the device's manifold system or ina mixing chamber upstream from the manifold. After introduction into thewhole blood, the lipid-based capture particles begin to bind to theparathion and/or paraoxon in the whole blood.

Next, the method 900 continues with the lipid-based capture particlesand the blood factors of the whole blood, which are, originally,substantially-evenly dispersed throughout the whole blood, beingdirected away from the walls of the separation channel (step 903) andaggregated into alignment at about the center of the separation channel.In some implementations, such as the implementation in the network 300as described above, this is done with a first bulk transducer 302imparting a standing acoustic wave across the channel transverse to thedirection of flow within the channel in an upstream portion 308,resulting in a pressure node at about the center of the separationchannel. In such implementations the contrast factor of the lipid-basedcapture particles has the same sign as that of the blood factors, thusthe blood factors and lipid-based capture particles move in tandemtowards the pressure node. This initial aggregation of particles along acommon axis enables later separation of the lipid-based captureparticles from the blood factors of blood due based on theirdifferential acoustophoretic mobility.

As discussed above in reference to network 300, after an initialaggregation (step 903), the method 900 continues with the lipid-basedcapture particles and blood factors of blood being directed alongside adownstream wall of the separation channel (step 904). In someimplementations, such as that of network 300, this is accomplished by ashift in the separation channel such that the downstream portion of theseparation channel is significantly aligned with the middle of theupstream portion of the separation channel.

As depicted in FIG. 3 above, in the downstream portion 309, the method900 continues with the blood factors being driven way from the walls ofthe separation channel (step 905). As mentioned above, in someimplementations, the contrast factor of the blood factors and thelipid-based capture particles have the same sign but are of differentmagnitudes. Thus, the blood factors will migrate away from the wall at afaster rate than the lipid-based capture particles and organophosphates,e.g., parathion and/or paraoxon. In other implementations, thelipid-based capture particles are designed to have a contrast factormagnitude larger than the blood factors of blood, thus the lipid-basedcapture particles move away from the wall at a faster rate than theblood factors.

In some implementations, the standing waves applied to the upstreamportion 308 and/or to the downstream portion 309 are periodically haltedfor a limited amount of time. Doing so allows lipid-based captureparticles or blood factors that may have become trapped against asidewall of the separation channel 300 to be released, therebypreventing clogging or congestion in the channel. For example, fordevices utilizing an excitation frequency between about 1.0-3.04, thestanding waves may be halted about once every second for about one tenthof second. In other implementations, the standing waves may be haltedmore or less frequently with shorter or longer durations depending, forexample, on the length and width of the channel and the flow rate offluid through the channel. In some implementations, the standing wavehas a duty cycle of between about 75% and about 98%, or between about78% and about 95%, between about 81% and about 92%, between about 84%and about 89%.

The method 900 concludes when the blood factors are collected in a firstdownstream outlet (step 906) and the lipid-based capture particles beingcollected in a second downstream outlet (step 907). As described abovein relation to network 300, the second downstream outlet 306 isconfigured to collect fluid containing lipid-based capture particlessubstantially devoid of blood factors. In some implementations, this isachieved by configuring the width of the second downstream outlet 306 tobe slightly less than d(f,x), the distance the blood factors travel inresponse to the standing acoustic wave given a flow rate off and adistance x from the point of application of the standing acoustic wave.Thus the blood factors, having been driven d(f,x) away from theseparating channel will be collected in the first downstream outlet 307.

FIG. 10 is a flow chart of a method for cleansing blood with asingle-stage microfluidic separation channel (1000). First, whole bloodis collected (step 1001). Then whole blood is flowed into an inlet of asingle-stage microfluidic separation channel, as depicted in FIG. 2(step 1002). Next, a plurality of lipid-based capture particles isintroduced into the whole blood (step 1003). Then a standing acousticwave is applied to the separation channel (step 1004). The blood factorsare then collected in a first outlet (step 1005). Next, the lipid-basedcapture particles are collected in a second outlet (step 1006). Finally,the cleansed blood is returned to a storage container or returneddirectly to the patient (step 1007).

Referring to FIGS. 1, 2 and 10, the method 1000 of cleansing blood witha single-stage microfluidic separation channel 200 begins by collectingwhole blood. In some implementations, the whole blood is collected froma patient 101, and then directly introduced into the blood cleansingsystem 100. In other implementations, the whole blood is collected froma patient 101 and then stored for later cleansing.

Next, the method 1000 of cleansing blood with a single-stagemicrofluidic separation channel 200 continues by flowing whole bloodinto the inlet of a microfluidic separation channel (step 1002). Thewhole blood contains a blood factors, plasma, and OP, e.g., parathionand/or paraoxon. In some implementations, a single microfluidicseparation channel is used, while in others a plurality of single-stageseparation channels is used in conjunction to accommodate greater bloodflow throughput.

The method 1000 continues with the introduction of a plurality oflipid-based capture particles into the whole blood (step 1003). In someimplementations, the constituent components of lipid-based captureparticles are injected into a separation channel with a micronozzle andspontaneously form lipid-based capture particles as injected into theseparation channel. In other implementations, the lipid-based captureparticles are prefabricated and then introduced into the whole blood. Insome implementations, the lipid-based capture particles are introducedinto the whole blood after the whole blood enters the separation channelthrough the first inlet 202. In yet other implementations, thelipid-based capture particles are introduced into the whole blood beforethe blood enters through the first inlet 202 of the separation channel200. In some implementations, the lipid-based capture particles aremicrobeads and/or lipid based liposomes.

Next, the method 1000 continues with the applying of a standing acousticwave to the separation channel (step 1004). The standing acoustic waveis applied transverse to a direction of flow of the whole blood throughthe separation channel 200. In some implementations, the blood factorsand lipid-based capture particles have contrast factors with differentsigns. Thus, the application of the standing acoustic wave causes theblood factors to aggregate about the central axis of the separationchannel and the lipid-based capture particles to aggregate along atleast one wall of the separation channel, as depicted in FIG. 2. Inother implementations the standing acoustic wave causes the bloodfactors to aggregate along at least one wall of the separation channeland the lipid-based capture particles to aggregate about the centralaxis of the separation channel.

In some implementations, the standing wave is periodically halted for alimited amount of time. Doing so allows lipid-based capture particles orblood factors that may have become trapped against a sidewall of theseparation channel 200 to be released, thereby preventing clogging orcongestion in the channel. For example, for devices utilizing anexcitation frequency between about 1.0-4.0 MHz, the standing wave may behalted about once every second for about one tenth of second. In otherimplementations, the standing wave may be halted more or less frequentlyor for shorter or longer durations depending, for example, on the lengthand width of the channel and the flow rate of fluid through the channel.In some implementations, the standing wave has a duty cycle of betweenabout 75% and about 98%, or between about 78% and about 95%, betweenabout 81% and about 92%, between about 84% and about 89%.

Then, the method 1000 continues with the collecting of the blood factorsof the whole blood in a first outlet (step 1005). In someimplementations, as depicted in FIG. 2, a first outlet 204 is alignedwith the central axis of the separation channel allowing the outlet tocollect the blood factors as they aggregate and flow down the centralaxis of the separation channel. Similarly, the method continues with thecollecting of the lipid-based capture particles in a second outlet (step1006). In some implementations, the end of the separation channel has atleast a second outlet channel 206 and 207 aligned with at least one wallof the separation channel. As the lipid-based capture particles aredriven towards the anti-pressure nodes along the walls of the separationchannel, they are collected by the outlets channels 206 and 207 alignedwith the walls of the separation channels. In some implementations, thestanding acoustic wave is adjusted such that the formed particle alignalong the walls of the separation channel and the lipid-based captureparticles align with the central axis of the separation channel. In suchan implementation, the blood factors are funneled into outlets along thewall of the separation channel and the lipid-based capture particles arefunneled into an outlet aligned with the central axis of the separationchannel. In some implementations, the outlet channels 206 and 207terminate in individual outlets or merge to terminate into a singleoutlet 205.

The method 1000 concludes with the reintroduction of the cleansed bloodinto a patient 101 or storage (step 1007). In some implementations, suchas system 100, the whole blood is collected directly from a patient andthen reintroduced to the patient 101. In some implementations, thecleansed blood is reheated to body temperature before being reintroducedinto the patient 101. In other implementations, the cleansed blood iscollected in a storage container for later reintroduction into a patient101.

EXAMPLES

The present examples are non-limiting implementations of the use of thepresent technology.

Example 1. Preparation of Lipid-Based Capture Particles

Provided below is a non-limiting exemplary method for making alipid-based capture particle in the form of a liposome.

Materials

Pyrromethene dye (Exciton #597-8C9), 1000 cSt Silicone oil (SigmaAldrich #378399),1,2-dilauroyl-sn-Glycero-3-[phosphor-rac-(1-glycerol)](Sodium Salt)(Avanti #840435P)/(Matreya, #1443), 1,2-dilauroyl-sn-glycero-3phosphocholine (Avanti #850702P) 18G syringe Liposofast-Basic (Avestin),PBS, 10 μm track etched membrane (Whatman #7060-4715), BChE (Protexia™),and PEG.

Method for Linking BChE to a Lipid Via PEG Linkers

BChE is linked to PEG-lipids, by dissolution of an activatedfunctionalized lipid (such as DSPE-PEG-NH₂) in an organic solvent suchas 1:2 ratio of DMSO/methanol followed by addition of BChE and couplingreagent EDC. The extent of the reaction is monitored by thin layerchromatography (TLC) using KMnO4 or p-anisaldehyde. The un-reactedlipid-PEG is extracted by gel electrophoresis.

Method for Making a Lipid-Based Particle Displaying BChE in the Form ofa Liposome

Emulsions are prepared through a multistep emulsification process bycombining fluorescent DLPG-silicone oil with PBS. The oil mixture isprepared by dissolving positively charged DLPG (Matreya, 1443) andpyrromethene dye (Exciton, 597-8C9) into 1000 cSt silicone oil (SigmaAldrich, 378399) for a final concentration of 0.5 mg/mL and 0.25 mg/mL,respectively. BChE-PEG-lipid is added to the oil mixture. Lipids and dyeis allowed to equilibrate in the oil for at least 24 hours. Using an 18Gsyringe, the oil is then aspirated up and down through an equal volumeof PBS, resulting in an aqueous phase and an oil phase. The aqueousphase is extracted with a larger 16G needle and loaded into one syringeof the liposofast (Avestin). A 10 μm track etched membrane (Whatman,7060-4715) is inserted between the mesh bearings of the liposofast andlocked into place. The emulsions are then extruded through the membraneby pushing the syringes of the liposofast back and forth 10 times.Lipid-based particles are analyzed with MatLAB.

In some implementations, the lipid-based particles produced above aredialyzed to remove small liposomes. Large pore dialysis is performed toremove liposomes less than 3 μm from the extruded liposomes. Dialysiscassettes (Thermo Scientific, 66810) are modified by replacing theoriginal membrane with a 3 μm filtration membrane (Whatman, 111712).Membranes are attached with Dow Corning 3140 and allowed to cure at 65°C. for at least 12 hours. Freshly extruded liposomes are then added tothe cassette and submerged into a beaker of PBS at a 1:100 ratio ofliposome solution to PBS. The beaker was placed on a magnetic stirrerwith stir-bar for 24 hours. After 24 hours, the solution containingliposomes greater than 3 μm was removed.

Example 2. Method for Clearing Parathion and/or Paraoxon from Guinea PigWhole Blood

Samples of guinea pig blood are saturated with excess paraoxon.Lipid-based capture particles are added to the contaminated blood, bloodwithout lipid-based capture particle treatment, and blood withoutparaoxon, are used as controls. The contaminated blood and controls areincubated at 37° C. for one hour. The contaminated blood and control areseparately passed through a microfluidic device as described above.Samples are collected from the blood outlet stream and are processed andanalyzed for free paraoxon concentration. The blood samples are analyzedfor BChE activity according to the Ellman assay and compared tountreated control. These data are used to determine OP clearanceefficiency and capacity of the filtration unit.

It is anticipated that the collected blood from the lipid-based captureparticle treated sample will have less free paraoxon than the untreatedblood sample, and will be comparable in purity to the control (noparaoxon). The results will indicated that the lipid-based captureparticles of the present technology are useful for removing parathionand/or paraoxon from parathion contaminated blood.

EQUIVALENTS

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended as singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatuses within the scope of theinvention, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled. It is to be understood that thisinvention is not limited to particular methods, reagents, compoundscompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

What is claimed:
 1. A device for removing organophosphates from wholeblood comprising: a microfluidic separation channel having an upstreamend and downstream end, the microfluidic separation channel comprising:a first inlet configured to introduce flowing whole blood into aproximal end of the microfluidic separation channel; a first outlet atthe downstream end of the microfluidic separation channel positionedsubstantially along a longitudinal axis of the microfluidic separationchannel; a second outlet at the downstream end positioned adjacent afirst wall of the microfluidic separation channel; and an acoustictransducer positioned adjacent to the microfluidic separation channelfor imposing a standing acoustic wave transverse to a flow of bloodthrough a particle migration region of the microfluidic separationchannel; and a lipid-based capture particle injector configured tointroduce lipid-based capture particles into the microfluidic separationchannel, wherein the lipid-based capture particles comprise silicone oiland organophosphate affinity molecules linked to a first population oflipids.
 2. The device of claim 1, wherein the lipid-based captureparticles further comprise a second population of lipids, wherein thesecond population of lipids form a lipid layer in which the firstpopulation of lipids is embedded.
 3. The device of claim 1, wherein thelipid-based capture particles are in the form of a liposome, vesicle,emulsion, lipid encapsulated droplet, or combinations thereof.
 4. Thedevice of claim 1, wherein the organophosphate affinity molecules areBChE.
 5. The device of claim 1, wherein the organophosphate affinitymolecules are linked to the first population of lipids with a PEGmolecule.
 6. The device of claim 1, wherein the silicone oil isencapsulated within the lipid-based capture particles.
 7. The device ofclaim 1, wherein the lipid-based capture particle injector is configuredto inject the lipid-based capture particles into the first inlet beforea particle migration region of the microfluidic separation channel. 8.The device of claim 1, wherein the microfluidic separation channelcomprises walls having a thickness at a particle aggregation point thatis greater than a multiple of one quarter of a wavelength of an acousticwave acting on the walls of the microfluidic separation channel.
 9. Amethod of cleansing blood of a subject comprising: flowing whole bloodinto an inlet of a microfluidic separation channel wherein the wholeblood comprises plasma and blood factors; introducing lipid-basedcapture particles into the whole blood, wherein the lipid-based captureparticles comprise silicone oil and organophosphate affinity moleculeslinked to a first population of lipids; and applying a standing acousticwave transverse to a direction of flow of the whole blood through themicrofluidic separation channel such that the blood factors aggregate toabout an axial center of the microfluidic separation channel and thelipid-based capture particles with bound organophosphates aggregatealong at least one wall of the microfluidic separation channel.
 10. Themethod of claim 9, further comprising cycling off the standing acousticwave such that a duty cycle of the standing acoustic wave is betweenabout 75% and about 95%.
 11. The method of claim 9, further comprisingcollecting blood factors of the whole blood at a first outlet positionedat a downstream end of the microfluidic separation channel at about theaxial center of the microfluidic separation channel.
 12. The method ofclaim 9, further comprising collecting lipid-based capture particlesthrough at least a second outlet positioned at a downstream end of themicrofluidic separation channel adjacent to the at least one wall alongwhich the lipid-based capture particles are aggregated.
 13. The methodof claim 9, wherein the lipid-based capture particles further comprisesa second population of lipids, wherein the second population of lipidsform a lipid layer in which the first population of lipids is embedded.14. The method of claim 9, wherein the lipid-based capture particleshave an opposite contrast factor than that of the blood factors.
 15. Themethod of claim 9, wherein the lipid-based capture particles are betweenabout 10 μm and 20 μm in diameter.
 16. A composition comprisingorganophosphate affinity molecules, a first population of lipids andsilicone oil, wherein the organophosphate affinity molecules are linkedto the first population of lipids, wherein the first population oflipids form a lipid-based capture particle, wherein the silicone oil isencapsulated within the lipid-based capture particle, and theorganophosphate affinity molecules are displayed on the surface of thelipid-based capture particle.
 17. The composition of claim 16, furthercomprising a second population of lipids, wherein the second populationof lipids form a lipid layer in which the first population of lipids isembedded.
 18. The composition of claim 16, wherein the organophosphateaffinity molecules are BChE.
 19. The composition of claim 16, whereinthe first population of lipids is selected from DSPE, DPPE, DMPE, or acombination thereof.
 20. The composition of claim 17, wherein the secondpopulation of lipids is selected from DOPC, DOPG, DOPE, or a combinationthereof.